Adjuvants are agents incorporated into vaccine formulations to enhance the immunogenicity of vaccine antigens. Aluminum salts (such as aluminum phosphate and aluminum hydroxide) are the most commonly used adjuvants used in human and veterinary vaccines today. While a number of aluminum containing adjuvants are available, for any one specific vaccine formulation, adjuvant/antigen effects provided by one may not be optimal.
Two methods have commonly been used to prepare vaccines and toxoids with aluminum compounds—in situ precipitation of aluminum compounds in the presence of antigen and adsorption of antigen onto preformed aluminum gel. Adsorption of antigens on aluminum, adjuvants, either during in situ precipitation of aluminum adjuvants or onto preformed aluminum gels, is dependant on the physical and chemical characteristics of the antigen, the type of aluminum adjuvant used and the conditions of adsorption. Factors which may affect an antigen's adsorption onto an aluminum adjuvant include electrostatic forces, hydrophobic interactions, Van der Waals forces, hydrogen binding, pH, temperature, size of gel particles, and the ionic strength of reaction mixture. In general, antigens are adsorbed to aluminum adjuvants through electrostatic attraction (i.e., adjuvant and antigen have opposite charges) and/or ligand exchange (e.g., phosphate group on antigen displaces a hydroxyl group on the adjuvant surface) (Seeber S J, et al Vaccine 1991; 9:201-3; Iyer S. et al, Vaccine 2004; 29:1475-9).
Aluminum hydroxide in its dehydrogenated, crystalline form is chemically aluminum oxyhydroxide [AlO(OH)] and in its aqueous phase, it becomes aluminum trihydroxide [Al(OH)3] by acquiring an additional water molecule (Hem S. L. et al 2007 Vaccine 25:4985-4986). Aluminum oxyhydroxide has a point of zero charge (PZC) of 11 and as such, is positively charged at pH 7.4. This positive charge makes aluminum oxyhydroxide a good adsorbent for negatively charged antigens (e.g. acidic proteins).
In one study, pretreatment of aluminum hydroxide adjuvant with phosphate anion was found to alter the surface charge characteristics of the adjuvant so that a basic protein (lysozyme, i.e. p.+11.1) could be adsorbed. The phosphate anion was found to reduce the adjuvant's positive zeta (ζ) potential (mV) and this alteration of the surface charge of the adjuvant changed the electrostatic forces between the adjuvant and lysozyme from repulsive to attractive such that the protein was adsorbed by the adjuvant (Rinella Jr. J. V., et al., Vaccine 1996; 14(no.4):298-300).
The maximum amount of antigen that can be adsorbed as a monolayer to the adjuvant is referred to as the “adsorptive capacity” and the strength of the adsorption force is called the “adsorptive coefficient” (Jendrick et al, Vaccine 2003; 21:3011-8). Studies of the effect of adsorptive capacity on vaccine immunogenicity suggest that the percentage of the antigen dose adsorbed is unrelated to a formulation's immunogenicity (Chang M-F. et al., Vaccine 2001;19:2884-9; Romero Mendez I Z et al Vaccine 2007; 25(5):825-33). In contrast, one study has shown a correlation between the adsorptive coefficient of an antigen to an aluminum containing adjuvant and the immune response elicited by the formulation (Hansen et al., Vaccine 2007; 25:6618-6624).
Adsorption may affect a protein's structure and stability. Results from studies on the effect of adsorption to aluminum containing adjuvants are not entirely consistent: in one, three proteins (bovine serum albumin (BSA), lysozyme and ovalbumin) were destabilized following adsorption onto Alhydrogel® or Adju-Phos®; in another study, the structure of BSA and Î2-lactoglobuline (BLG) was stabilized by adsorption onto aluminum hydroxide (Jones L. S. et al., J. Biol Chem 2005; 280(14):13406-13414; Zheng Y. et al., Spectroscopy 2007;21(5-6):257-268). Methods for stabilizing for storage liquid formulations of vaccine compositions with aluminum salt adjuvants include lypohilization, freezing and freeze-drying, but often result in adjuvant agglomeration, decreased immunogen concentration and loss of immunogenicity (e.g., Maa et al, (2003) J. Pharm. Sci. 92:319-332; Diminsky et al. (1999) Vaccine 18:3-17; Alving et al (1993) Ann. NY Acad. Sci. 690:265-275; and Warren et al (1986) Ann Rev Immunol. 4:369-388, all of which are incorporated by reference). Even for those formulations maintained under refrigerated conditions (e.g. 2° C. to 8° C.) adsorbed antigens may be chemically unstable and as such, over time may under go hydrolysis and fragmentation. Therefore, a process for the production of a vaccine composition comprising an aluminum salt adjuvant that addresses these issues (e.g., chemical instability, decrease in antigen concentration) is needed.